WO1998044100A1 - Dissociation de molecules reagissant mutuellement - Google Patents

Dissociation de molecules reagissant mutuellement Download PDF

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Publication number
WO1998044100A1
WO1998044100A1 PCT/GB1998/000975 GB9800975W WO9844100A1 WO 1998044100 A1 WO1998044100 A1 WO 1998044100A1 GB 9800975 W GB9800975 W GB 9800975W WO 9844100 A1 WO9844100 A1 WO 9844100A1
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Prior art keywords
voltage
disassociation
molecules
nucleic acid
buffer
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PCT/GB1998/000975
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English (en)
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David Nicholas Miller-Jones
Karin Bergmann
Susan Louise Watson
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Scientific Generics Limited
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Application filed by Scientific Generics Limited filed Critical Scientific Generics Limited
Priority to EP98913960A priority Critical patent/EP0973885B1/fr
Priority to CA002286302A priority patent/CA2286302A1/fr
Priority to DE69827375T priority patent/DE69827375T2/de
Priority to AT98913960T priority patent/ATE281517T1/de
Priority to JP54131598A priority patent/JP4004548B2/ja
Priority to US09/381,896 priority patent/US6333157B1/en
Priority to KR10-1999-7008981A priority patent/KR100510606B1/ko
Publication of WO1998044100A1 publication Critical patent/WO1998044100A1/fr
Priority to HK00104385A priority patent/HK1025129A1/xx

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Definitions

  • This invention relates to processes for the treatment of interacting molecules in order to effect a complete or partial disassociation thereof.
  • Double-stranded DNA (deoxyribonucleic acid) and DNA/RNA ( ribonucleic acid) and RNA/RNA complexes in the familiar double helical configuration are produced by the stable interaction of single-stranded molecules.
  • Such complexes in vitro require aggressive conditions to separate the complementary strands of the nucleic acid.
  • Known methods that are commonly employed for strand separation require the use of high temperatures of at least 60°C and often 100°C or use an alkaline pH of 11 or higher or a low pH .
  • helicase enzymes such as Rep protein of E.coli that can catalyse the unwinding of the DNA in an unknown way, or binding proteins such as 32 -protein of E.coli phage T4 that act to stabilise the single-stranded form of DNA.
  • the denatured single-stranded DNA produced by the known processes of heat or alkali treatment is used commonly for hybridisation studies or is subjected to amplification cycles. Such separation is a prerequisite of a number of protocols involving the in vitro manipulation of nucleic acids, one example of which is a reaction that produces multiple copies of target sequences of DNA and which employs a heat-stable polymerase enzyme (US Patent No. 4683202, K.B. Mullis et al) .
  • WO 92/04470 discloses a process whereby nucleic acid strands are separated by the application of an electric field. The advantages of the electrical method are discussed in greater detail, along with the method's application in ampli- fication reactions such as PCR and ligase chain reaction. Forms of electrochemical cells for carrying out the reaction are described and also the use of "promoter" compounds that enhance the efficiency of denaturation.
  • a PCR reaction is conducted in which there are repeated denaturation operations conducted using the electrochemical cell described with intervening amplification stages.
  • the denaturation stages are each conducted for a period of five minutes or longer and the total time for the PCR reaction is therefore very extended.
  • the conditions under which the PCR reaction was conducted in O92/00470 differ from those of the conventional PCR process in that it was not found possible to use a conventional PCR buffer system. In order to obtain denaturation, it was necessary to conduct the process at a much lower ionic strength than would be consistent with such a buffer system. Excluding the promoter methyl viologen, the process was basically conducted in distilled water.
  • the concentration of said promoter cation is preferably from 1 Mm to 50 Mm, more preferably from 5 Mm to 20 Mm, e.g. about 10 Mm.
  • the rate and extent of denaturation obtainable in such electrochemical systems depends on a number of factors, including the medium in which the nucleic acid is present. Processes used in molecular biology such as nucleic acid hybridisation assays or amplification procedures like PCR are conducted in media containing a buffering agent to maintain optimum Ph for the reactions involved. However, the presence of such a buffering agent is generally adverse to the electrochemical dehybridisation of nucleic acids. This is to some extent overcome by an appropriate choice of promoter, as described above, but it would be highly desirable to develop systems in which the presence of the buffer was substantially less adverse in its impact on the dehybridisation process .
  • disassociation permissive buffers allow disassociation to proceed without the addition of disassociation promoting agents such as magnesium or lithium ions, or viologens .
  • Tris-HCl is an example of a buffer of the type which may be used when suitable promoters are present . As is shown below, the buffers now proposed are superior in their ability to permit disassociation to proceed when compared with Tris- HCl.
  • the buffers may be used to permit the disassociation of other interacting molecules, especially biomolecules, under the influence of an electrical voltage.
  • the type of interaction between the molecules may in particular be hydrogen bonding.
  • the mechanism by which the buffers used according to this invention permit or encourage disassociation is not at present fully understood. It may be that disassociation is caused by a local change in pH in the solution in which the electrodes are immersed, such a change occurring in a microlayer adjacent the electrode surface and producing acid conditions at the positive anode and alkaline conditions at the negative cathode. This would be consistent with the fact that DNA can be caused to denature by both acid and alkaline pH. Buffers permitting the temporary and local generation of a relatively low pH, i.e. those which themselves have a pK which is relatively high may allow the necessary pH change to occur when other buffers having a lower pK a would prevent it.
  • Buffers show maximum buffering capacity when the pH of the solution is the same as the pK a of the buffer: as the pH moves away from the p a , buffering capacity is reduced. Therefore, a solution of CHES (pK a 9.4) at pH 7.5 - 8.0 has weak buffering capacity, and its pH is readily deflected downwards .
  • CHES pK a 9.4
  • pH 7.5 - 8.0 has weak buffering capacity, and its pH is readily deflected downwards .
  • the medium stabilises to its former pH due to the action of the buffering agent .
  • This ability of the buffer to reversibly "flip" pH in response to an electric field may be related to its pK value.
  • the buffer may be that it is the intercalation of the buffer into the nucleic acid double helix which is responsible for encouraging or permitting denaturation of double- stranded nucleic acids and that by virtue of either the spacing between the charges or the presence of the cyclohexyl ring, the preferred buffers described herein are particularly suited to use in such systems. A combination of these mechanisms may be acting simultaneously.
  • the present invention provides a process for disassociating interacting molecules, comprising subjecting a liquid containing said molecules to an electrical voltage applied between electrodes under conditions such as to wholly or partially disassociate at least a proportion of said molecules in the presence of an electrical disassociation permissive buffer which is more disassociation permissive than Tris-HCl.
  • the molecules may be biomolecules and in particular may be nucleic acid molecules associated by hybridisation (so that said disassociation constitutes denaturation of said hybridised nucleic acid molecules) or one of said interacting biomolecules may be an immunological binding partner of the other of said biomolecules.
  • one of said inter- acting biomolecules may be a ligand and the other may be a receptor for said ligand.
  • the molecules may be macromolecules, one or both of which may be charged, as in a DNA duplex for example.
  • Said buffer preferably has a pK a of not less than 8.5, more preferably not less than 9.0, most preferably not less than 9.2.
  • the process is preferably conducted at a pH of from 7 to 9.
  • the buffer chosen will to some extent dictate the pH at which the disassociation reaction can be performed.
  • the pH may be selected to optimise the activity of the enzyme.
  • the choice of buffer and of enzyme will be related.
  • the buffer CHES is particularly preferred for use with "Vent" polymerase, but buffers with higher pK a 's such as CAPS or CAPSO may be a more appropriate choice with enzymes with higher pH optima such as Bst DNA polymerase from Bacillus stearothermophilus (pH 8-9) , "Deep Vent” (New England Biolabs) from Pyrococcus sp (pH 8.8) , Dynazyme II DNA polymerase (Finnzymes Oy) from Ther us brockianus (pH 8.5), Taq DNA polymerase and derivatives from Thermus aqua ticus (pH 8.8), or T4 DNA polymerase from bacteriophage T4 (pH 8.8).
  • Bst DNA polymerase from Bacillus stearothermophilus (pH 8-9)
  • "Deep Vent” New England Biolabs
  • Dynazyme II DNA polymerase Feinnzymes Oy
  • Ther us brockianus pH
  • the buffer is preferably CHES, CAPS or CAPSO.
  • the pK a values of these buffers are quoted in the literature either as working values or as back corrected thermodynamic values, which are a little higher. In relation to the preferred ranges of pK quoted above, regard should be had to the thermodynamic values which are: CHES 9.41, CAPS 10.51 and CAPSO 9.71.
  • the buffer comprises a molecule having a negative charge separated from a positive charge by a distance of from 0.75 to 1.5 times the distance between such charges in CHES, CAPS or CAPSO molecules.
  • the buffer has a moiety able to intercalate into double-stranded nucleic acids, e.g. a cyclohexyl substituent. The process is preferably conducted in from a 5 to 10 mM concentration of said buffer.
  • the nucleic acid or other molecule does not have to be dissolved in the solution containing the buffer but may be immobilised to a solid phase' immersed in the solution.
  • the single-stranded nucleic acid or other released molecule produced is free from the electrode, e.g. dissolved in the solution.
  • a nucleic acid may be immobilised on the electrode or another surface in the cell or a coating on the electrode in double or single-stranded form prior to the application of the electric potential, e.g.
  • the portion of the nucleic acid by which it is attached is preferably one selected for the purpose by the user .
  • a reference electrode may be contacted with said solution and a voltage may be applied between said electrode and said counter-electrode so as to achieve a desired controlled voltage between said electrode and said reference electrode.
  • the electrodes may be connected by a potentiostat circuit as is known in the electrochemical art.
  • a potential of from -0.5 to -1.5 V is applied to said working electrode with respect to said reference electrode, more preferably from -0.8 to -1.1 V, e.g. about - 1.0 V.
  • Working electrode voltages relative to reference elec- trodes are given throughout as if measured or as actually measured relative to a calomel reference electrode (BDH No. 309.1030.02) .
  • the process may be conducted using a three electrode system of the kind described in WO92/04470 but generally it is preferred that the volume of solution employed according to this invention is small e.g. 1 ml or less, preferably very small e.g. 100 ⁇ l or less, e.g. about 25 ⁇ l to 40 ⁇ l .
  • the volume of solution employed according to this invention is small e.g. 1 ml or less, preferably very small e.g. 100 ⁇ l or less, e.g. about 25 ⁇ l to 40 ⁇ l .
  • a voltage will be applied between two electrodes and will be measured directly. Voltages given herein for two electrode systems are given in this way and not with reference to a calomel electrode.
  • a further alternative preferred form of cell comprises a pair of platinum plate electrodes separated by an elastomer sheet containing a cut out defining the cell cavity.
  • the elastomer may have a thickness of from 100 ⁇ m to 1 mm, more preferably from 200 ⁇ m to 800 ⁇ m, most preferably from 300 ⁇ m to 500 ⁇ m.
  • the process is conducted at a voltage of 1.5 to 2.5 volts measured as a voltage difference between the electrodes.
  • the applied voltage will generally need to be increased to compensate for the voltage drop across the coating.
  • a constant current regime it will generally be preferable to use a current of from 80 to 160 ⁇ A, e.g. about 100 to 125 ⁇ A.
  • a promoter compound such as methyl viologen as described in WO92/04470 to produce more rapid disassociation or denaturation.
  • Other promoters are described in W093/15224, i.e. multivalent cations such as magnesium.
  • multivalent cations which are effective and which can be used include lanthanum (La 3 + ) .
  • the cations used as the promoters may include inorganic cations complexed with inorganic or organic ligands, e.g. Pt(NH 3 ) g 4+ and Cr(NH 3 ) g 2+ .
  • a promoter may be any inorganic or organic molecule which increases the rate or extent of denaturation of the double helix. It should be soluble in the chosen reaction medium. It preferably does not affect or interfere with DNA or other materials such as enzymes or oligonucleotide probes which may be present in the solution. Alternatively, the promoter may be immobilised to or included in material from which the electrode is constructed.
  • the additional promoter may be a water-soluble compound of the bipyridyl series, especially a viologen such as methyl - viologen or a salt thereof. Whilst the mechanism of operation of such promoters is presently not known with certainty, it is believed that the positively charged viologen molecules interact between the negatively charged nucleic acid such as DNA and the negatively charged cathode to reduce electrostatic repulsion therebetween and hence to promote the approach of the DNA to the electrode surface where the electrical field is at its strongest. Accordingly, one preferred option is to employ as promoters compounds having spaced positively charged centres, e.g. bipolar positively charged compounds. Preferably the spacing between the positively charged centres is similar to that in viologens .
  • Suitable viologens include ethyl-viologen, isopropyl -viologen and benzyl- viologen.
  • the ionic strength of said solution is preferably no more than 250 mM, more preferably no more than 100 mM . As it has been found that the rate of denaturation increases as the ionic strength is decreased, the said ionic strength is still more preferably no more than 50 mM, e.g. no more than 25 mM or even no more than 5 mM. Generally, the lower the ionic strength, the more rapid is the denaturation. However, in calculating ionic strength for these purposes it may be appropriate to ignore the contribution to ionic strength of any component which acts as a promoter as described above.
  • the electrode may be a so called "modified electrode” in which the denaturing is promoted by a compound coated on to, or adsorbed on to, or incorporated into the structure of the electrode which is otherwise of an inert but conducting material .
  • a first preferred form of electrochemical cell for use in this invention is described below which uses a carbon rod electrode dipping into a carbon block containing a well.
  • working, counter and optionally reference electrodes may be formed on a single surface, e.g. a flat surface by any printing method such as thick film screen printing, ink jet printing, or by using a photo-resist followed by etching. It is also possible that the counter and reference electrodes can be combined on the flat surface leading to a two electrode configuration.
  • the electrodes may be formed on the inside surface of a well which is adapted to hold liquid, such a well could be the well known 96 well or Microtitre plate, it may also be a test tube or other vessel. Electrode arrays in Microtitre plates or other moulded or thermoformed plastic materials may be provided for multiple nucleic acid denatura- tion experiments or other disassociation reactions. The reactin may be carried out on a damp porous member, e.g. filter paper.
  • Nucleic acid strand separation may be carried out in an aqueous medium or in a mixture of water with an organic solvent such as dimethylformamide .
  • the use of polar solvents other than water or non-polar solvents is also accepted but is not preferred.
  • the process may be carried out at ambient temperatures or if desired temperatures up to adjacent the pre-melting temperature of the nucleic acid.
  • the denaturing process carried out on nucleic acids according to the invention may be incorporated as a step in a number of more complex processes, e.g. procedures involving the analysis and/or the amplification of nucleic acid. Some examples of such applications are described below.
  • the invention includes a process for detecting the presence or absence of a predetermined nucleic acid sequence in a sample which comprises: denaturing a sample of double- stranded nucleic acid by means of an electrode; hybridising the denatured nucleic acid with an oligonucleotide probe for the sequence; and determining whether the said hybridisation has occurred, wherein during denaturation the solution contains a said buffer.
  • the invented process has application n DNA and RNA hybridisation where a specific gene sequence is to be indent1- fied e.g. specific to a particular organism or specific to a particular hereditary disease of which sickle cell anaemia is an example.
  • a specific sequence is to be indent1- fied e.g. specific to a particular organism or specific to a particular hereditary disease of which sickle cell anaemia is an example.
  • a sample of DNA preferably of purified DNA, means for which are known, which is in native double-stranded form. It is then necessary to convert the double-stranded DNA to single-stranded form before a hybridisation step with a labelled nucleotide probe which has a complementary sequence to the DNA sample can take place.
  • the denaturation process of the invention can be used for this purpose in a preferred manner by carrying out the following steps: denaturing a sample of DNA by applying a voltage by means of an electrode to the sample DNA m contact with a said buffer in solution; hybridising the denatured DNA with a directly labelled or indirectly labelled nucleotide probe complementary to the sequence of interest; and determining whether the hybridisation has occurred, which determination may be by detecting the presence of the probe, the probe being directly radio-labelled, fluorescent labelled, chemilummescent labelled or enzyme- labelled or being an indirectly labelled probe which carries biotin for example to which a labelled avidin or avidm type molecule can be bound later.
  • a typical DNA probe assay it is customary to immobilise the sample DNA to a membrane surface which may be composed of neutral or charged nylon or nitrocellulose.
  • the immobilisation is achieved by charge interactions or by baking the membrane containing DNA m an oven.
  • the sample DNA can be heated to high temperature to ensure conversion to single- stranded form before binding to the membrane or it can be treated with alkali once on the membrane to ensure conversion to the single-stranded form.
  • the disadvantages of the present methods are : heating to high temperatures to create single-stranded
  • DNA can cause damage to the sample DNA itself; - the use of alkali requires an additional step of neutralisation before hybridisation with the labelled probe can take place.
  • One improved method for carrying out DNA probe hybridisation assays is the so called "sandwich" technique where a specific oligonucleotide is immobilised on a surface.
  • the surface having the specific oligonucleotide thereon is then hybridised with a solution containing the target DNA in a single-stranded form, after which a second labelled oligonucleotide is then added which also hybridises to the target DNA.
  • the surface is then washed to remove unbound labelled oligonucleotide, after which any label which has become bound to target DNA on the surface can be detected later.
  • This procedure can be simplified by using the disassociation process of the invention to denature the double-stranded DNA into the required single-stranded DNA.
  • the working electrode, counter electrode and optionally a reference electrode and/or the promoter can be incorporated into a test tube or a well in which the DNA probe assay is to be carried out.
  • the DNA sample, promoter if not already present and oligonucleotide probes can then be added and the voltage applied to denature the DNA.
  • the resulting single-stranded DNA is hybridised with the specific oligonucleotide immobilised on the surface after which the remaining stages of a sandwich assay are carried out. All the above steps can take place without a need for high temperatures or addition of alkali reagents as in the conventional process.
  • the electrochemical denaturation of DNA can be used in the amplification of nucleic acids, e.g. in a polymerase chain reaction, ligase chain reaction amplification procedure or a strand displacement amplification technique.
  • the present invention provides a process for replicating a nucleic acid which comprises: separating the strands of a sample double- stranded nucleic acid in contact with or dissolved in a solution containing a said buffer under the influence of an electrical voltage applied to the solution from an electrode; hybridising the separated strands of the nucleic acid with at least one oligonucleotide primer that hybridises with at least one of the strands of the denatured nucleic acid; synthesising an extension product of an or each primer which is sufficiently complementary to the respective strand of the nucleic acid to hybridise therewith; and separating the or each extension product from the nucleic acid strand with which it is hybridised to obtain the extension product.
  • the said amplification process further comprises repeating the procedure defined above cyclicly, e.g. for more than 10 cycles, e.g. up to 20 or 30 cycles.
  • the hybridisation step is preferably carried out using two primers which are complementary to different strands of the nucleic acid.
  • the denaturation to obtain the extension products as well as the original denaturing of the target nucleic acid is preferably carried out by applying to the solution of the nucleic acid a voltage between electrodes, the solution con- taining a buffer as described herein.
  • the process may be a standard or classical PCR process for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids wherein each nucleic acid consists of two separate comple- mentary strands, of equal or unequal length, which process comprises :
  • step (c) treating the single-stranded molecules generated from step (b) with the primers of step (a) under conditions such that a primer extension product is synthesised using each of the single strands produced in step (b) as a template.
  • the process may be any variant of the classical or standard PCR process, e.g. the so-called “inverted” or “inverse” PCR process or the "anchored” PCR process .
  • the invention therefore includes an amplification process as described above in which a primer is hybridised to a circular nucleic acid and is extended to form a duplex which is denatured by the denaturing process of the invention, the a plification process optionally being repeated through one or more additional cycles.
  • the invention includes a process for replicating a target sequence of nucleic acid comprising hybridisation, extension and denaturation of nucleic acid (e.g. cycles of hybridising and denaturing) wherein said denaturation is produced by operating on a solution containing said nucleic acid with an electrode in the presence of a said buffer .
  • the process of the invention is applicable to the ligase chain reaction.
  • the invention includes a process for amplifying a target nucleic acid comprising the steps of: (a) providing nucleic acid of a sample as single-stranded nucleic acid; (b) providing in the sample at least four nucleic acid probes, wherein: i) the first and second of said probes are primary probes, and the third and fourth of said probes are secondary nucleic acid probes; ii) the first probe is a single strand capable of hybridising to a first segment of a primary strand of the target nucleic acid; iii) the second probe is a single strand capable of hybridising to a second segment of said primary strand of the target nucleic acid; iv) the 5' end of the first segment of said primary strand of the target is positioned relative to the 3' end of the second segment of said primary strand of the target to enable joining of the 3' end of the first probe to the 5' end of the second probe, when said probes are hybridised to said primary strand of said target nucle
  • the denaturation of the DNA to allow subsequent hybridisation with the primers can be carried out by the application of an appropriate potential to the electrode.
  • the process may be carried out stepwise involving successive cycles of denaturation or renaturation as in the existing thermal methods of PCR and LCR, but it is also possible for it to be carried out continuously since the process of chain extension or ligation by the enzyme and subsequent strand separation by the electrochemical process can continue in the same reaction as nucleic acid molecules in single-stranded form will be free to hybri- dise with primers once they leave the denaturing influence of the electrode.
  • the electrochemical DNA amplification technique can be used analytically to detect and analyse a very small sample of DNA e.g. a single copy gene in an animal cell or a single cell of a bacterium.
  • the time required for denaturation to occur may be extremely short, e.g. less than 0.5 second up to 1.0 second.
  • a process of repeated denaturation of double- stranded nucleic acid may be performed, in which said voltage is applied as a repeating pulse having a duration of up to 2 minutes, e.g. up to one minute or much less.
  • the voltages may be turned off and/or reversed for a period similar to or equal to the period for which the voltage is applied, e.g. the voltage may be applied as pulses at a frequency of from 0.01 to 10 Hz.
  • a single denaturation may be performed using a single pulse cycle.
  • the voltage may be applied such that there are, in other any order, periods of application of voltage with a first polarity, periods of application of voltage with the opposite polarity to said first polarity and periods of substantially reduced applied voltage.
  • the cycles may be from 0.01 seconds to 5 minutes or more in length, e.g. from 1 second to 5 minutes in length.
  • the periods during which said voltage is applied with a first polarity and said periods during which said voltage is applied with a second polarity are each independently from 0.5 seconds to 1 minute.
  • the periods during which said voltage is substantially reduced are each independently from 0.5 seconds to 3 minutes.
  • the invention includes a kit for use in a process of disassociating interacting molecules which kit comprises, an electrode, a counter electrode and optionally a reference electrode, and a said buffer.
  • the kit may further include any or all of one or more oligonucleotide probes, an enzyme such as polymerase, one or more primers, or a disassociation promoter, e.g. a source of lithium ions.
  • the probe if present, may be labelled in any of the ways discussed above.
  • the reassociation of molecules, particularly the rehybri- disation of nucleic acid strands may be produced or promoted by the application of a reverse voltage using a similar buffer and other conditions as described herein in relation to disassociation.
  • Figure 1 is an exploded view diagram of an electrochemical cell used for denaturation of DNA.
  • Figure 2 shows a time/voltage profile suitable for use in operation of the cell of Figure 1.
  • Figure 3 shows gels obtained in Example 1; and Figure 4 shows a gel obtained in Example 2.
  • the cell shown in Figure 1 comprises a sandwich formed between a pair of opposed glass plates 10, 11 outside a pair of opposed platinum foil electrodes 12, 13 which lie either side of a spacer sheet of a silicone elastomer (silasticTM) .
  • silicone elastomer silicone elastomer
  • a pair of chambers 15, 16 are formed in the silasticTM sheet.
  • Each chamber consists of a circular cut-out of diameter approximately 10 mm and a lead-in channel extending to the edge of the sheet.
  • the silasticTM sheet is approximately 400 ⁇ m in thickness.
  • Each electrode is provided with a connecting tag 17, 18 by which electrical connection is made to it.
  • illustrated electrochemical cell comprises two opposing planar platinum electrodes.
  • the electrodes are separated by a sheet of deformable insulating material (in this instance a silicone elastomer) which forms a seal against liquid loss, and which is cut to form the electrode chamber.
  • the electrodes are backed by flat plates, and the whole is clamped together between aluminium blocks (not shown) comprising the elastomer sheet from a free state thickness of about 500 ⁇ m.
  • the potential difference between the electrodes and the polarity reversal pattern over time are set on a PC, which controls a power supply.
  • the electrode assemblies are maintained at the operating temperature (suitably 55 °C) by standing them on a heating block of appropriate temperature.
  • electrical denaturation is performed in the electrochemical cell by applying a potential between the electrodes.
  • 45 ⁇ l of linearised pUC 18 plasmid (2868 base pairs) at 0.5 ⁇ g/ml in water or buffer is placed in the cell.
  • a potential difference is applied across the electrodes for a fixed time period, with or without a number of polarity changes.
  • the sample is then removed from the cell and subjected to agarose gel electro- phoresis. Denaturation is judged to have occurred when a downward shift in the position of the band is observed.
  • Figure 3 shows three gels containing pUC DNA in some of the buffers tested, after exposure to the electrical profile in Figure 2.

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Abstract

L'invention concerne des molécules d'ADN double brin et d'autres molécules qui réagissent mutuellement, par exemple un anticorps et un antigène, qui sont dissociées par application d'une tension électrique à une solution contenant lesdites molécules et ce, dans un tampon favorisant ladite dissociation, le tampon étant par exemple CHES, CAPS ou CAPSO.
PCT/GB1998/000975 1997-04-02 1998-04-02 Dissociation de molecules reagissant mutuellement WO1998044100A1 (fr)

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EP98913960A EP0973885B1 (fr) 1997-04-02 1998-04-02 Dissociation de molecules reagissant mutuellement
CA002286302A CA2286302A1 (fr) 1997-04-02 1998-04-02 Dissociation de molecules reagissant mutuellement
DE69827375T DE69827375T2 (de) 1997-04-02 1998-04-02 Dissoziation von interagierenden molekülen
AT98913960T ATE281517T1 (de) 1997-04-02 1998-04-02 Dissoziation von interagierenden molekülen
JP54131598A JP4004548B2 (ja) 1997-04-02 1998-04-02 相互作用する分子の解離
US09/381,896 US6333157B1 (en) 1997-04-02 1998-04-02 Disassociation of interacting molecules
KR10-1999-7008981A KR100510606B1 (ko) 1997-04-02 1998-04-02 상호작용 분자의 해리 방법
HK00104385A HK1025129A1 (en) 1997-04-02 2000-07-18 Disassociation of interacting molecules.

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GBGB9706654.2A GB9706654D0 (en) 1997-04-02 1997-04-02 Disassociation of interacting molecules
GB9706654.2 1997-04-02

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EP (1) EP0973885B1 (fr)
JP (1) JP4004548B2 (fr)
KR (1) KR100510606B1 (fr)
AT (1) ATE281517T1 (fr)
CA (1) CA2286302A1 (fr)
DE (1) DE69827375T2 (fr)
GB (1) GB9706654D0 (fr)
HK (1) HK1025129A1 (fr)
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001055708A2 (fr) * 2000-01-27 2001-08-02 Abbott Laboratories Procede permettant de traiter un echantillon qui contient au moins un element biologique
WO2004018081A1 (fr) * 2002-08-21 2004-03-04 Tokyo University Of Agriculture And Technology Tlo Co., Ltd. Procede de purification d'une substance biologique avec un support d'affinite cassable par voie electrolytique, support d'affinite cassable par voie electrolytique, trousse pour purification de substance biologique et lieur cassable par voie electrolytique

Families Citing this family (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69534386T2 (de) * 1995-08-25 2006-04-20 Affymetrix, Inc. (a Delaware Corp.), Santa Clara Freigabe von intrazellulärem material
US20050069926A1 (en) * 2003-08-01 2005-03-31 Affymetrix, Inc. Helicase-amplified reverse transcription
KR101214179B1 (ko) * 2003-08-06 2013-01-10 더 프록터 앤드 갬블 컴퍼니 표적 물질 검출용 가교 부재
NZ551996A (en) * 2004-05-13 2010-07-30 Anita Goel Nano-PCR: Methods and devices for nucleic acid amplification and detection
US7888125B2 (en) 2005-05-09 2011-02-15 Theranos, Inc. Calibration of fluidic devices
US11287421B2 (en) 2006-03-24 2022-03-29 Labrador Diagnostics Llc Systems and methods of sample processing and fluid control in a fluidic system
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US11286519B2 (en) 2013-12-11 2022-03-29 Accuragen Holdings Limited Methods and compositions for enrichment of amplification products
WO2016168755A1 (fr) 2015-04-17 2016-10-20 Distributed Bio, Inc. Procédé pour l'humanisation de masse d'anticorps non humains
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992004470A1 (fr) * 1990-09-12 1992-03-19 Scientific Generics Limited Denaturation electrochimique d'acide nucleique bicatenaire
EP0541238A1 (fr) * 1991-10-07 1993-05-12 Northeastern University Colonne capillaire pour la séparation électrophorétique à haute performance et la détection de protéines SDS et système utilisant cette colonne
US5213669A (en) * 1992-01-31 1993-05-25 Beckman Instruments, Inc. Capillary column containing a dynamically cross-linked composition and method of use
WO1993015224A1 (fr) * 1992-01-23 1993-08-05 Scientific Generics Limited Traitement electrochimique de solutions contenant de l'acide nucleique
WO1995025177A1 (fr) * 1994-03-15 1995-09-21 Scientific Generics Limited Dénaturation électro-chimique de l'acide nucléique double brins
WO1997008293A1 (fr) * 1995-08-25 1997-03-06 Scientific Generics Limited Liberation de matiere intracellulaire
WO1998000562A1 (fr) * 1996-07-02 1998-01-08 Scientific Generics Limited Denaturation d'acide nucleique double brin
WO1998002573A1 (fr) * 1996-07-11 1998-01-22 Scientific Generics Limited Denaturation de l'acide nucleique a double brin

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5935825A (en) * 1994-11-18 1999-08-10 Shimadzu Corporation Process and reagent for amplifying nucleic acid sequences

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992004470A1 (fr) * 1990-09-12 1992-03-19 Scientific Generics Limited Denaturation electrochimique d'acide nucleique bicatenaire
EP0541238A1 (fr) * 1991-10-07 1993-05-12 Northeastern University Colonne capillaire pour la séparation électrophorétique à haute performance et la détection de protéines SDS et système utilisant cette colonne
WO1993015224A1 (fr) * 1992-01-23 1993-08-05 Scientific Generics Limited Traitement electrochimique de solutions contenant de l'acide nucleique
US5213669A (en) * 1992-01-31 1993-05-25 Beckman Instruments, Inc. Capillary column containing a dynamically cross-linked composition and method of use
WO1995025177A1 (fr) * 1994-03-15 1995-09-21 Scientific Generics Limited Dénaturation électro-chimique de l'acide nucléique double brins
WO1997008293A1 (fr) * 1995-08-25 1997-03-06 Scientific Generics Limited Liberation de matiere intracellulaire
WO1998000562A1 (fr) * 1996-07-02 1998-01-08 Scientific Generics Limited Denaturation d'acide nucleique double brin
WO1998002573A1 (fr) * 1996-07-11 1998-01-22 Scientific Generics Limited Denaturation de l'acide nucleique a double brin

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001055708A2 (fr) * 2000-01-27 2001-08-02 Abbott Laboratories Procede permettant de traiter un echantillon qui contient au moins un element biologique
WO2001055708A3 (fr) * 2000-01-27 2002-05-02 Abbott Lab Procede permettant de traiter un echantillon qui contient au moins un element biologique
JP2003530087A (ja) * 2000-01-27 2003-10-14 アボット・ラボラトリーズ 少なくとも１種の生物学的要素を含む試料を処理する方法
JP2015070840A (ja) * 2000-01-27 2015-04-16 アボツト・モレキユラー・インコーポレイテツド 少なくとも１種の生物学的要素を含む試料を処理する方法
WO2004018081A1 (fr) * 2002-08-21 2004-03-04 Tokyo University Of Agriculture And Technology Tlo Co., Ltd. Procede de purification d'une substance biologique avec un support d'affinite cassable par voie electrolytique, support d'affinite cassable par voie electrolytique, trousse pour purification de substance biologique et lieur cassable par voie electrolytique

Also Published As

Publication number Publication date
HK1025129A1 (en) 2000-11-03
US6333157B1 (en) 2001-12-25
JP4004548B2 (ja) 2007-11-07
EP0973885A1 (fr) 2000-01-26
CA2286302A1 (fr) 1998-10-08
DE69827375D1 (de) 2004-12-09
DE69827375T2 (de) 2005-10-20
ATE281517T1 (de) 2004-11-15
KR20010005904A (ko) 2001-01-15
EP0973885B1 (fr) 2004-11-03
JP2001519658A (ja) 2001-10-23
GB9706654D0 (en) 1997-05-21
KR100510606B1 (ko) 2005-08-31

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